Article Figures & Data
Figures
- Fig. 1.
Microglial infection. Human adult microglial cultures were established as previously described (1, 2, 95, 116) and infected with 5 ng/ml p24gag of supernatants containing env-pseudotyped, luciferase viruses. After 48 to 62 h, cells were lysed and the extent of infection was measured by the intensity of chemiluminescence when mixing equal volumes of substrate and cell lysate. A representative experiment (performed in triplicate) is shown, and the results are expressed as the mean + standard deviation (error bars).
- Fig. 2.
Effect of sCD4 on infection of cells expressing CCR5 alone or with CD4. (A) HOS cells stably transfected for the expression of CD4 (HOS-CD4), CCR5 (HOS-CCR5), or both (HOS-CD4-CCR5) were infected with supernatants containing env-pseudotyped, luciferase viruses (p24gag [5 ng/ml]). The results are expressed as the mean + standard deviation (error bars) from four independent experiments each performed in triplicate. (B) Treatment with sCD4 (5 μg/ml) inhibited infection of HOS-CD4-CCR5 cells by rB15 and rV1V2 but not by rBORI pseudotypes. Results are shown as percent luciferase activity relative to the value in the absence of sCD4 treatment, in four independent experiments each performed in triplicate. The differences in values between rBORI and rB15 or rV1V2 were statistically significant (Wilcoxon's rank-sum test: P < 0.05 in both cases).
- Fig. 3.
Infection of cells expressing decreasing amounts of CD4 and/or CCR5. U87 cells were transiently transfected with plasmids expressing CD4 and CCR5 and subsequently infected with supernatants containing env-pseudotyped, luciferase viruses (p24gag [5 ng/ml]). (A) rBORI pseudotype did not infect cells expressing low CD4 and high CCR5 or low CD4/CCR5, while rB15 and rV1V2 did. Results are expressed as the mean + standard deviation (error bars) from seven independent experiments each performed in triplicate. (B) The luciferase activity measured following infection of low-CD4 and high-CCR5 or low-CD4/CCR5 cells was expressed as a percentage of the activity induced in the high-CD4/CCR5 cells. We performed seven independent experiments with the rBORI, rB15, and rV1V2 pseudotypes. The differences in the values between rBORI and rB15 or rV1V2 were statistically significant (Wilcoxon's rank-sum test: P < 0.01 in both comparisons for low CD4 and high CCR5 and for low CD4/CCR5).
- Fig. 4.
Infection of cells expressing CD4 and the 5552 or Δ4 chimeric coreceptors. U87 cells were transiently transfected with plasmids expressing CD4 and the chimeric coreceptors 5552 (amino terminus, ECL1 and ECL2 of CCR5, and ECL3 of CCR2b) or Δ4 (deletion of the first four amino acids in the amino terminus of CCR5), and subsequently infected with supernatants containing env-pseudotyped, luciferase viruses (p24gag [5 ng/ml]). Luciferase activity is shown as the percentage with respect to wild-type CCR5 for each virus and experiment. Horizontal lines represent the median of each group of data. For the 5552 chimera, rBORI infections were significantly less efficient than rB15 and rV1V2 infections (Wilcoxon's rank-sum test: P < 0.001 in both cases). For the Δ4 mutant, rBORI infected significantly less than rB15 and rV1V2 ( P < 0.01 and P < 0.02 , respectively).
- Fig. 5.
Neutralization with HIV-1-positive human sera. A MAGI-CCR5 cell-based, single-cycle infection assay was performed as described in Materials and Methods, to test the neutralization sensitivity of VH-rBORI, VH-rB15, and VH-rV1V2 recombinant viruses (93) either in the presence of a 1/10 dilution of NHS or in the presence of increasing dilutions of serum from four HIV-1-infected individuals. Results were calculated as percentage of infection with respect to the extent observed with virus plus NHS, and the values shown represent the average of two to three independent experiments.
- Fig. 6.
17b ELISA and neutralization assays. (A) An ELISA was performed using the 17b MAb with supernatants containing env pseudotypes, either with or without sCD4. Results are shown as mean optical density at 405 nm + standard error of the mean (error bars) from four independent experiments using different pseudotype stocks. Absent sCD4, reactivity with the 17b antibody was greater in rB15 and rV1V2 than in rBORI pseudotypes (Student's t test: P < 0.02 and P < 0.05 , respectively). (B) 17b MAb neutralization was performed in U87 cells transiently transfected with CD4-CCR5 (left; four independent experiments) and in microglial cultures (right; three independent experiments with two microglial preparations). The pseudotypes were incubated with 17b MAb (20 μg/ml) at 37°C for 1 h prior to inoculation. Data are shown as percentage of luciferase activity with respect to untreated controls for each pseudotype. In both cell types, 17b inhibited infection by rB15 and rV1V2 pseudotypes but not by rBORI.
Tables
- Table 1.
Amino acid differences and syncytium-inducing phenotype, in the context of whole or recombinant viruses, of envused for the production of pseudotypes
env-pseudotyped virus Amino acid differences with respect to BORI or rBORI No. of potential N-linked glycosylation sites lost Syncytium-inducing phenotype No. Amino acid change(s) and positiona BORI 0 0 Noninducing rBORI 0 0 Noninducing B15 8 E153G, T162Ab, R166G, S190Rb, K229N, T281I, A336T, V532M 2 High rB15 8 E153G, T162Ab, R166G, S190Rb, K229N, T281I, A336T, V532M 2 High rV1V2 4 E153G, T162Ab, R166G, S190Rb 2 High rE153G 1 E153G 0 High rE153G,T162A 2 E153G, T162Ab 1 Noninducing a Numbers are based on the HXB2 sequence. BORI and B15 contain HXB3 sequences at both the carboxy and amino termini, as discussed in Materials and Methods and by Shieh et al. (93).
b Change results in the loss of a potential N-linked glycosylation site.
- Table 2.
Infection of U87 cells expressing CD4 and the indicated chimeric coreceptor by env-pseudotyped, luciferase viruses
Coreceptor Mean luciferase activity ± SD (RLU/s)a rBORI rB15 RV1V2 rE153G rE153G, T162A None 132 ± 68 195 ± 112 281 ± 161 141 ± 64 149 ± 59 CCR5 b 24,611 ± 11,755 75,090 ± 15,932 108,548 ± 25,902 42,948 ± 16,214 38,309 ± 15,001 CCR2b 98 ± 35 187 ± 137 250 ± 70 192 ± 84 171 ± 64 5222 643 ± 590 1,509 ± 1,225 2,413 ± 2,356 1,414 ± 769 938 ± 1,356 2522 74 ± 39 79 ± 38 119 ± 55 60 ± 48 72 ± 51 2252 71 ± 33 75 ± 41 79 ± 11 54 ± 22 72 ± 33 2225 101 ± 61 107 ± 55 269 ± 216 86 ± 37 77 ± 21 2555 735 ± 904 614 ± 513 792 ± 573 208 ± 226 361 ± 356 5255 8,604 ± 4,539 28,211 ± 13,617 49,532 ± 14,623 13,489 ± 4,553 12,803 ± 6,802 5525 587 ± 399 2,284 ± 2,169 4,519 ± 2,191 2,101 ± 1,485 688 ± 593 5552 1,071 ± 814 15,960 ± 6,144 26,984 ± 8,637 5,845 ± 2,832 4,231 ± 3,273 5252 2,912 ± 1,269 9,104 ± 3,599 19,053 ± 11,073 3,892 ± 1,620 7,640 ± 4,246 Δ4 1,734 ± 1,025 12,501 ± 8,077 18,904 ± 11,742 2,731 ± 3,092 4,250 ± 3,338 Δ8 1,545 ± 1,023 2,442 ± 1,990 3,780 ± 2,227 3,362 ± 1,849 1,435 ± 1,603 Δ12 134 ± 112 91 ± 38 63 ± 18 62 ± 17 59 ± 21 Δ16 122 ± 108 89 ± 41 49 ± 22 47 ± 26 65 ± 26 a Luciferase activity is calculated from 5 to 17 independent experiments performed at least in triplicate.
b Values in bold represent statistical significant changes in comparison with wild-type CCR5, also in bold, as indicated in the text.
